To control viral infections the host has developed an integrated defense network that comprises both innate and adaptive immune responses. Initial responses to viral infection involve primarily the innate response and the killing of infected cells with cytotoxicity. Once the virus has invaded the cell, host-mediated responses are triggered by single stranded and doubled stranded viral RNAs that induce expression of stress cytokines including interferons (IFNs). IFNs are essential for initiating and coordinating a successful antiviral response by inducing a number of intracellular genes, the IFN-stimulated genes (ISGs), which prevent virus replication/cytolysis and stimulate the adaptive arm of the immune system. Cellular mechanisms that prevent viral replication, dissemination or persistent infections include global inhibition of protein synthesis, blockage of protein transport and induction of apoptosis. A number of cellular proteins have been implicated as mediators of virus-induced apoptosis, including two of the proteins that were the focus of our original proposal, TRAIL/Apo-2L and X-linked inhibitor of apoptosis-associated factor-1 (XAF1). IFNs also potently regulate cellular responsiveness to virus and other cellular pathogens by upregulating components of the virus detection system, including toll-like receptors, RIG-I and downstream signaling molecules. Recently, we have shown that XAF1 augments cellular sensitivity to apoptotic agents such as TRAIL, and can also enhance cellular antiviral responses. XAF1 is shown herein to impact signaling cascades initiated by TNF family ligands and dsRNA signaling cascades. This grant is designed to functionally dissect the mechanism of XAF1 activity. Emphasis will be on downstream regulators of XAF1 activity, including a newly identified protein, ZNF313, which we now show to be a XAF1-binding RING finger protein that is post-translationally regulated by IFNs. The role of this putative E3 ubiquitin ligase in regulating IFN, viral and other innate immune responses will be pursued as outlined in the following aims. We will also pursue recent data linking XAF1 to other signaling molecules that regulate ubiquitin ligation, including TRAF2 and RIP2, and demonstrate that TRIP6, another XAF1 binding partner, may represent a novel ubiquitination regulator in the TNF/dsRNA pathway.